Activation of Multiple Signal Transduction Pathways as a Prognostic Marker in Acute Myelogenous Leukemia.

Abstract

Deregulation of signal transduction pathways (STPs) including JAK/STAT, RAS/Raf/MEK/ERK and PI3K/AKT may promote leukemogenesis by conferring cells proliferation and survival advantages in acute myelogenous leukemia (AML). The activation of these pathways had an adverse prognosis in AML and development of targeted therapies seems to be promising. Heat-shock proteins (HSP) are involved in the conformational maturation of a number of signaling proteins, and HSPs expression in AML is associated with other adverse prognostic factors (Bcl2, MRP). The aim of this work was to study STPs expression in AML, and there correlation to other adverse prognostic factors and complete remission rate. Sixty five patients with primary AML were analyzed by flow cytometry for constitutive ERK, PI3K and AKT activation, HSP90, Bcl2 and P170 expression. FAB subtypes were M0=3, M1=19, M2=16, M4=10, M5=16, M6=1. All patients received an induction treatment, 44 patients reached complete remission. Cytogenetics was available in 63 cases (Intermediate = 36, favorable = 3, unfavorable= 24). We performed a 3 color flow cytometry protocol using CD45 and CD34 to identify blast cells, and used a third antibody to the following transduction proteins (ERK, pERK, AKT, pAKT, PI3K) or intracytoplasmic proteins (Bcl2, HSP90, p170). Spontaneous growth of leukemic progenitor cells (CFU-L) was investigated in 44 samples. In AML, activated proteins were found in CD34+ cells. ERK and PI3K/AKT were frequently co-activated. Flow cytometry results showed that levels of STPs were significantly higher (p<0,05) in patients who did not reach complete remission, with a shorter survival: PI3K (12% of positive cells for CR patients vs 68% for non CR patients), ERK (17% vs 69%), pERK (15% vs 77%), AKT (24% vs 76%) and pAKT (10% vs 79%). All patients who reached complete remission had a percentage of positive cells < 20% (except 2 cases for AKT). Interestingly, the levels did not differ significantly according to cytogenetics, and were only marginally higher in patients with high WBC, which suggests an independent prognostic value. By contrast, we found a good correlation between activation of signaling pathways and HSP90, Bcl2 and p170 expression (p<0,05). We observed a spontaneous growth in 30 cases (defined by more than 10 colonies/105cells), with a good correlation with HSP90, Bcl2, p170 and CD34 expression. By contrast, there was no correlation between spontaneous growth and activation of STPs, suggesting that spontaneous growth of leukemic cells which is also a prognostic factor is not directly related to activation of STPs. These results indicate that the activation of multiple STPs is common in AML and is associated with poor prognosis, and that flow cytometry technique can be used for rapid detection of AML patients who are resistant to conventional chemotherapy. This is of particular interest because of development of targeted therapies for signaling transduction pathways, chaperones and anti-apoptotic proteins.